The authors would like to thank Dr. Charles Luetje for providing the T-maze and the behavior room. This work is supported by the American Heart Association (to Y.A.), the National Institute for Neurological Disorder and Stroke Grant R01NS64269 (to R.G.Z.), and the Pew Charitable Trust (to R.G.Z.).

1. 	Introduction
In this video, we will demonstrate two behavioral assays using Drosophila to demonstrate the measurement of locomotion and learning and memory functions that are compromised in neurodegenerative disorders. First, we will show how locomotor behavior can be measured using the negative geotaxis assay. Second, we will assess learning and memory abilities using positively-phototactic flies that are trained to associate light with an aversive bitter taste and then tested 6 hours later to assess memory functions. Finally, we will discuss the implications and limitations of these behavior assays.
2. 	Equipment and Reagents
<ol>
<li>Fly work
<ul>
<li>Stereo scope (Zeiss)</li>
<li>Small paint brushes for pushing flies</li>
<li>Drosophila carbon dioxide (CO2) anesthesia apparatus (Geneseesci, Inc)</li>
</ul>
</li>
<li>Negative Geotaxis Assay
<ul>
<li>28.5 x 95 mm Polystyrene Vial (Capitol Vial, Inc.)</li>
<li>Mini-Alarm Timer/Stopwatch (VWR International) </li>
<li>Sharpie pens</li>
<li>Adhesive Tape</li>
</ul>
</li>
<li>Aversive Phototaxis Suppression Assay
<ul>
<li>Quinine Hydrochloride (Sigma Aldrich; CAS Number: 6119-47-7)</li>
<li>Distilled Water</li>
<li>Digital measuring scale for precise dilution by weight</li>
<li>Quick Disconnect Connectors, High-Density Polyethylene (NALGENE VWR Catalog No. 62868-021)</li>
<li>15 mL Centrifuge Tube (VWR International), cut at the 2 mL mark from closed end to fit with the Quick Disconnect Connector adaptor for light source </li>
<li>Single-channel 200 &mu;L pipetter to load quinine solution onto filter paper lining the chamber</li>
<li>T-Maze (Simple Behavioral Systems; Figure 1)</li>
<li>Aluminum Foil</li>
<li>Light Source (Zeiss)</li>
<li>A lamp with red light.</li>
<li>Filter Paper</li>
<li>Mini-Alarm Timer/Stopwatch (VWR International)</li>
</ul>
</li>
</ol>
3. 	Preparing the Flies
<ul>
<li>Flies are maintained on standard cornmeal-agar-molasses-yeast medium at 25&deg;C on a 12 hr light/dark cycle. </li>
<li>Virgin flies are isolated under carbon dioxide anesthesia on the day of eclosion. For aging studies, flies are maintained in groups of twenty per vial and transferred to a fresh vial every 3 days. It is important to use sibling flies from the same parental cross for aging studies, to minimize differences arising from genetic backgrounds.</li>
<li>For both the assays, flies are sexed and maintained in groups of ten flies per vial. The gender difference on behavior is significant and it is advisable not to mix male and female flies in a study as data might be hard to interpret and reproduce6. </li>
<li>For negative geotaxis assay, flies are sorted into groups of ten per vial and tested one hour after anesthesia. It is important for flies to recover completely from anesthesia as this might have an effect on locomotor activities.</li>
<li>For APS assay, flies of each group are placed in an empty polystyrene vial with water moistened filter paper for six hours before the assay is performed. This ensures that the flies are starved before the assay and will be more perceptive to aversive taste. Just before the assay is performed, each fly from a group of ten is placed in an empty 15mL centrifuge tube, which is then capped loosely, to avoid escape. </li>
</ul> 
4. 	Negative Geotaxis Assay
<ul>
<li>Geotaxis is generally measured for ten to twenty groups of ten individuals of the same genotype or treatment (100-200 flies total for each genotype/treatment)7. Sort groups of ten female or male flies on a CO2 anesthesia apparatus and place each group in a separate vial. Allow at least one hour for the flies to recover from anesthesia.</li>
<li>Prepare the climbing apparatus for each group, such that two empty polystyrene vials are vertically joined by tape facing each other. It is important to make sure that the openings of the vials are perfectly aligned with each other to provide an even climbing surface for the flies. </li>
<li>For the lower vial, measure a vertical distance of 8 cm above the bottom surface and mark each vial by drawing a circle around the entire circumference of the vial.</li>
<li>Transfer a group of ten flies into the lower vial carefully preventing the escape of any fly. Immediately cover the lower vial with the top vial and tape securely near the contacting openings. Allow the flies to acclimatize to the new setting for 1 minute before conducting the assay.</li>
<li>Gently tap the flies down to the bottom of the vial and measure the number of flies that can climb above the 8-cm mark by 10 seconds after the tap.</li>
<li>Repeat this assay for the same group ten times, allowing for 1 minute rest period between each trial.</li>
<li>Record the number of flies per group that passed the 8-cm mark as a percentage of total flies. </li>
</ul>
5. 	Aversive Phototaxis Suppression Assay (APS Assay)
Originally adapted from Le Bourg and Buecher (2002)4, this assay exploits the positive phototactic behavior in flies to train them to associate light with aversive stimuli (in this case, bitter taste of quinine). After a training/conditioning phase, wild type flies will be able to associate the lighted area with aversive taste and avoid it. Flies with compromised learning capacities will fail to make this association. Furthermore, the APS assay can also be used to measure short term memory function of flies5 by subjecting already trained flies to the same test 6 hours post conditioning to test their ability in remembering the learned task.
<ol>
<li>Preparing the Quinine Solution
<ul>
<li>Dissolve quinine hydrochloride in distilled water to make a 0.1M stock solution (1.98g in 50mL distilled water). The stock solution can be kept at -20&deg;C for up to a year in small aliquots</li>
<li>Prepare a working solution of 1 &mu;M by diluting the stock solution in distilled water.</li>
</ul>
</li>
<li>Preparing the T-Maze
<ul>
<li>T-maze consists of the center column with the trap door and two independent chambers, a "dark" chamber and a "lighted" chamber (Figure 1). </li>
<li>Chamber preparation: take two 15mL plastic centrifuge tubes and cut at 2 mL mark from the bottom closed end with a manual saw. Fit the connector adapters at each end, and seal the connection with parafilm. The adapter at the end of the tube serves as the input slot for the gooseneck light source. Connect one tube with the light source and this tube will be the "lighted" chamber. Wrap the other tube with aluminum foil to serve as the "dark" chamber. </li>
<li>Add 180uL of either distilled water or quinine solution to filter paper and place in the light chamber.</li>
<li>Assemble the T-maze by screwing the lighted and dark chambers on each side of the center column, with the trap door in the middle closed (Figure 1). </li>
<li>Insert the light source into the lighted chamber. </li>
</ul>
</li>
<li>Training flies to suppress phototaxis with aversive stimuli
<ul>
<li>Unscrew the dark chamber from the T-maze and transfer a single fly into this chamber and immediately screw the dark tube back to the maze.</li>
<li>Turn off the lights in the room and turn on the red lamp.</li>
<li>Allow the fly to acclimatize in the dark chamber for 30 seconds and slowly turn on the light source illuminating the lighted chamber. Slowly open the trap door that separates the two chambers.</li>
<li>If the fly walks to the lighted chamber within 10 seconds, it is considered positively phototactic and is ready to be trained for the assay. Failure in phototaxis indicates problems in the visual system and flies with negative phototaxis need to be excluded from the assay. </li>
<li>For Aversive Phototaxis Suppression (APS) training, tap the fly that demonstrated positive phototaxis back to the dark chamber, close the trap door and turn off the light. Allow 30 seconds of acclimatizing. During this time put filter paper with quinine solution into the lighted chamber. Slowly open the trap door and turn on the light. Allow the fly walk into the quinine coated lighted chamber. After one minute, tap the fly back to the dark chamber and repeat this 9 more times. (Typically, wild type flies avoid the lighted chamber after 3 to 5 training trials.)</li>
<li>Immediately after training, five test trials will be conducted. In each test trial, allow 10 seconds after light is turned on for the trained fly to walk to the lighted chamber. Failure to walk to the lighted vial will be recorded as "Pass", which is equivalent to "task learned through reinforcement". The Pass rate over five consecutive trials is recorded and shown below in Figure 2 as PC0 (0 hr post conditioning).</li>
</ul>
</li>
<li>Assessing Short-Term Memory Function in Flies
<ul>
<li>After training and initial PC0 recordings, each fly is placed back into its original food vial and kept aside for six hours.</li>
<li>Six hours post training, subject each fly to 5 trials again in the same way as before, and record the number of times the fly avoids (pass) or goes into (fail) the lighted vial. This pass rate is recorded as PC6 (6 hrs post conditioning), which is an indicator of short term memory.</li>
</ul>
</li>
</ol>
6. 	Data Interpretation and Statistical Analysis
<ol>
<li>Negative Geotaxis Assay
<ul>
<li>The raw data generated from this assay represents the number of flies crossing the 8-cm mark in 10 seconds in each group. Convert this to percentage and compute the average pass rate for each group over 10 sessions.</li>
<li>Data is represented graphically as an average pass rate per group (genotype, treatment etc.) with the standard error of mean (SEM).</li>
<li>Differences between groups can then be determined to be statistically significant or not using a Student T-test or ANOVA.</li>
</ul>
</li>
<li>Aversive Phototaxis Suppression Assay
<ul>
<li>The raw data from this assay is represented as the number of times each fly avoids the lighted chamber in five successive trials. The pass rate is calculated as the percentage of successful avoidance trials over the total trials. Calculate the average pass rate in this manner for a minimum of 15 flies per group.</li>
<li>Data is graphically represented as average pass rate PC0 (immediately after conditioning; "learning" indicator) or average pass rate PC6 (6 hours post conditioning; "memory" indicator) with respective SEM.</li>
<li>Learning and memory indices are compared between groups with both wild type and "genetically-manipulated-benign" group (such as GFP overexpressing flies) appropriately.</li>
</ul>
</li>
</ol>
7. 	Representative Results
In this video experiment, we are assessing learning and memory and motor deficits in flies overexpressing human Tau in the central nervous system and comparing their performance to those of flies overexpressing GFP. Neuronal overexpression of human Tau in Drosophila has been shown to cause severe vacuolization in the brain8 and lead to significant deficits in learning and memory functions9. As shown in Figure 2A, 10 day old male or female flies overexpressing human Tau have significant locomotor deficits compared to age-matched GFP overexpressing flies. In addition, these flies failed to associate light with bitter taste as shown by PC0 pass rate compared to the control group, as well as fail to remember this association as shown by a decline in PC6 pass rate (Figure 2B). This experiment further validates that Drosophila models can be used for studying human neurodegenerative conditions and recapitulating some of the behavioral phenotypes observed in humans and mammals.
<img src="/files/ftp_upload/2504/2504fig1.jpg" alt="Figure 1" /> 
Figure 1. T-Maze Setup for Aversive Phototaxis Suppression Assay. A. The overall experimental setup for the APSA experiment with the light source connected to the "lighted" falcon tube, lined with filter paper and the "dark" tube on the left (covered in foil), separated by a trap door. B. During the training and testing phase, the fly is in the dark chamber and the trap door opened after the light is turned on in the lighted chamber.
<img src="/files/ftp_upload/2504/2504fig2.jpg" alt="Figure 2" /> 
Figure 2. Representative results from the negative geotaxis and APS experiment using flies overexpressing GFP or Tau with a pan-neuronal driver. A. In the negative geotaxis assay, 10-day old male (blue column) or female (red column) flies overexpressing GFP showed higher climbing activity than Tau-overexpressing flies. B. 20-day old female flies overexpressing GFP showed better learning (PC0) and memory (PC6) functions than Tau overexpressing flies.

<ol>
	<li>Bonini, N. M., Fortini, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+neurodegenerative+disease+modeling+using+Drosophila.">Human neurodegenerative disease modeling using Drosophila.</a> Annual Review of Neuroscience. 26, 627-656 (2003).</li><li>Lloyd, T. E. &. a. m. p. ;. a. m. p., Taylor, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flightless+flies:+Drosophila+models+of+neuromuscular+disease.">Flightless flies: Drosophila models of neuromuscular disease.</a> Ann N Y Acad Sci. 1184, e1-e20 (2010).</li><li>Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+mutants+of+Drosophila+isolated+by+countercurrent+distribution.">Behavioral mutants of Drosophila isolated by countercurrent distribution.</a> Proc. Natl. Acad. Sci. USA. 58, 1112-1119 (1967).</li><li>Le Bourg, E., Buecher, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learned+suppression+of+photopositive+tendencies+in+Drosophila+melanogaster.">Learned suppression of photopositive tendencies in Drosophila melanogaster.</a> Animal Learning &#38; Behavior. 30, 330-341 (2002).</li><li>Seugnet, L., Suzuki, Y., Stidd, R., Shaw, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aversive+phototaxic+suppression:+evaluation+of+a+short-term+memory+assay+in+Drosophila+melanogaster.">Aversive phototaxic suppression: evaluation of a short-term memory assay in Drosophila melanogaster.</a> Genes Brain Behav. 8, 377-389 (2009).</li><li>Nilsen, S. P., Chan, Y. -. B., Huber, R., Kravitz, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gender-selective+patterns+of+aggressive+behavior+in+Drosophila+melanogaster.">Gender-selective patterns of aggressive behavior in Drosophila melanogaster.</a> Proc. Natl. Acad. Sci. USA. 101, 12342-12347 (2004).</li><li>Leal, S. M., Neckameyer, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+evidence+for+GABAergic+regulation+of+specific+behaviors+in+Drosophila+melanogaster.">Pharmacological evidence for GABAergic regulation of specific behaviors in Drosophila melanogaster.</a> J Neurobiol. 50, 245-261 (2002).</li><li>Wittmann, C. W., Wszolek, M. F., Shulman, J. M., Salvaterra, P. M., Lewis, J., Hutton, M., Feany, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tauopathy+in+Drosophila:+Neurodegeneration+Without+Neurofibrillary+Tangles.">Tauopathy in Drosophila: Neurodegeneration Without Neurofibrillary Tangles.</a> Science. 293, 711-714 (2001).</li><li>Mershin, A., Pavlopoulos, E., Fitch, O., Braden, B. C., Nanopoulos, D. V., Skoulakis, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learning+and+memory+deficits+upon+TAU+accumulation+in+Drosophila+mushroom+body+neurons.">Learning and memory deficits upon TAU accumulation in Drosophila mushroom body neurons.</a> Learn Mem. 11, 277-287 (2004).</li><li>Ganetzky, B., Wu, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indirect+Suppression+Involving+Behavioral+Mutants+with+Altered+Nerve+Excitability+in+DROSOPHILA+MELANOGASTER.">Indirect Suppression Involving Behavioral Mutants with Altered Nerve Excitability in DROSOPHILA MELANOGASTER.</a> Genetics. 100, 597-614 (1982).</li><li>Hall, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetylcholinesterase+mutants+in+Drosophila+and+their+effects+on+the+structure+and+function+of+the+central+nervous+system.">Acetylcholinesterase mutants in Drosophila and their effects on the structure and function of the central nervous system.</a> J Comp Neurol. 189, 741-774 (1980).</li></ol>

No conflicts of interest declared.

Both negative geotaxis and APS assays are robust behavioral assays to measure changes in locomotor or learning and memory capacities arising from genetic or environmental manipulation. However, there are some disadvantages to these assays that await further optimization.
In negative geotaxis assay, mutations in certain genes can render severe motor dysfunctions in flies. In such cases, the flies can hardly climb the walls in response to gravity upon agitation. Other behavior assays that directly measure movement may be more appropriate. These assays include righting reflex behavior that measures paralysis and seizure10, and optomotor behavior11 that records loss of movement coordination.
In APS assay, the first hindrance can be flies randomly stopping at the entrance of the lighted chamber without moving forward. This becomes more of an issue if aging is one of the conditions studied, since older flies are less active in general and tend to stop and rest for long periods of time. In this case the experimenter has to provide mechanical stimuli to promote movement, which is not always effective. Second, one might argue that the failure to go to the lighted chamber results from a loss of positive phototaxis. This is rarely the case, since previous work has shown that flies turn back to light responsiveness once the negative reinforce is removed4. Third, the duration of contact with the aversive stimulus may vary from trial to trial. This variation can usually be reduced by encircling the inner wall of the chamber with soaked filter paper to prevent flies from avoiding the quinine. In addition, the experimenter can ensure that each fly spend enough time in contact with the filter paper by increasing the amount of time spent in the chamber from the otherwise noted 1 minute. 
In summary, negative geotaxis and APS assays are fast and simple ways of assessing behavioral deficits and quantitatively indicate locomotor and learning and memory abilities in Drosophila. These assays are particularly useful in modeling human neurodegenerative conditions in Drosophila.

<table border="1">
 <tbody>
 <tr>
 <th>Materials</th>
 <th>Company</th>
 <th>Catalog Number</th>
 <th>Comment</th>
 </tr>
 <tr>
 <td> Polystyrene Vial </td>
 <td> Capitol Vial, Inc. </td>
 <td> 28Fly </td>
 <td> 28.5 x 95 mm Polystyrene Vial; material quality important, bad if too slippery for flies to climb.</td>
 </tr>
 <tr>
 <td> Dissecting Stereoscope</td>
 <td> Zeiss</td>
 <td> 000000-1432-979 </td>
 <td> 2 branches goose-neck light guide (12V/30W) </td>
 </tr>
 <tr>
 <td> Quinine Hydrochloride</td>
 <td> Sigma Aldrich</td>
 <td> 6119-47-7</td>
 <td> Dissolve in distilled water.</td>
 </tr>
 <tr>
 <td> Quick Disconnect Connectors </td>
 <td> Nalgene (VWR)</td>
 <td> 62868-021</td>
 <td> High-Density Polyethylene</td>
 </tr>
 <tr>
 <td> T-Maze </td>
 <td> Simple Behavioral Systems</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td> Single-channel 200 &mu;L pipette </td>
 <td> Eppendorf</td>
 <td> 022443305</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

assaying locomotor, learning, and memory deficits in drosophila models of neurodegeneration

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of these diseases, including Alzheimer's, Parkinson's and Huntington's disease are characterized by age-dependent deterioration in learning and memory functions and movement coordination2. Here we use behavioral assays, including the negative geotaxis assay3 and the aversive phototaxic suppression assay (APS assay)4,5, to show that some of the behavior characteristics associated with human neurodegeneration can be recapitulated in flies. In the negative geotaxis assay, the natural tendency of flies to move against gravity when agitated is utilized to study genes or conditions that may hinder locomotor capacities. In the APS assay, the learning and memory functions are tested in positively-phototactic flies trained to associate light with aversive bitter taste and hence avoid this otherwise natural tendency to move toward light. Testing these trained flies 6 hours post-training is used to assess memory functions. Using these assays, the contribution of any genetic or environmental factors toward developing neurodegeneration can be easily studied in flies.

Watch this Scientific Journal Video about Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration at JoVE.com

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Behavior assays for measuring locomotor functions, learning, and memory abilities in Drosophila.

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of ...

assaying-locomotor-learning-memory-deficits-drosophila-models

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JoVE Journal

Neuroscience

33.4K Views.  University of Miami, Miller School of Medicine. Behavior assays for measuring locomotor functions, learning, and memory abilities in Drosophila.

Video: Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (&equiv;24 hr) pacemakers or clocks. Malfunctions in the human circadian system are associated with numerous diseases or disorders. Much progress towards our understanding of the mechanisms underlying circadian rhythms has emerged from genetic screens whereby an easily measured behavioral rhythm is used as a read-out of clock function. Studies using Drosophila have made seminal contributions to our understanding of the cellular and biochemical bases underlying circadian rhythms. The standard circadian behavioral read-out measured in Drosophila is locomotor activity. In general, the monitoring system involves specially designed devices that can measure the locomotor movement of Drosophila. These devices are housed in environmentally controlled incubators located in a darkroom and are based on using the interruption of a beam of infrared light to record the locomotor activity of individual flies contained inside small tubes. When measured over many days, Drosophila exhibit daily cycles of activity and inactivity, a behavioral rhythm that is governed by the animal's endogenous circadian system. The overall procedure has been simplified with the advent of commercially available locomotor activity monitoring devices and the development of software programs for data analysis. We use the system from Trikinetics Inc., which is the procedure described here and is currently the most popular system used worldwide. More recently, the same monitoring devices have been used to study sleep behavior in Drosophila. Because the daily wake-sleep cycles of many flies can be measured simultaneously and only 1 to 2 weeks worth of continuous locomotor activity data is usually sufficient, this system is ideal for large-scale screens to identify Drosophila manifesting altered circadian or sleep properties.

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

We describe procedures for recording daily locomotor activity rhythms of Drosophila and subsequent data analysis. Locomotor activity rhythms are a reliable behavioral output of animal circadian clocks and are used as the standard readout of clock function when studying circadian mutants or examining how the environment regulates the circadian system.

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (&equiv;24 hr) ...

Morris Water Maze Test for Learning and Memory Deficits in Alzheimer&#39;s Disease Model Mice

The Morris Water Maze (MWM) was first established by neuroscientist Richard G. Morris in 1981 in order to test hippocampal-dependent learning, including acquisition of spatial memoryand long-term spatial memory 1. The MWM is a relatively simple procedure typically consisting of six day trials, the main advantage being the differentiation between the spatial (hidden-platform) and non-spatial (visible platform) conditions 2-4. In addition, the MWM testing environment reduces odor trail interference 5. This has led the task to be used extensively in the study of the neurobiology and neuropharmacology of spatial learning and memory. The MWM plays an important role in the validation of rodent models for neurocognitive disorders such as Alzheimer&#x2019;s Disease 6, 7. In this protocol we discussed the typical procedure of MWM for testing learning and memory and data analysis commonly used in Alzheimer&#x2019;s disease transgenic model mice.

Morris Water Maze Test for Learning and Memory Deficits in Alzheimer&#039;s Disease Model Mice

The Morris Water Maze is a behavioral task to test hippocampal-dependent learning and memory. It has been widely used in the study of neurobiology, neuropharmacology and neurocognitive disorders in rodent models.

The Morris Water Maze (MWM) was first established by neuroscientist Richard G. Morris in 1981 in order to test hippocampal-dependent learning, including ...

Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also propagation of suprathreshold spiking activity involves populations of neurons. Empirical studies addressing cortical function directly thus require recordings from populations of neurons with high resolution. Here we describe an optical method and a deconvolution algorithm to record neural activity from up to 100 neurons with single-cell and single-spike resolution. This method relies on detection of the transient increases in intracellular somatic calcium concentration associated with suprathreshold electrical spikes (action potentials) in cortical neurons. High temporal resolution of the optical recordings is achieved by a fast random-access scanning technique using acousto-optical deflectors (AODs)1. Two-photon excitation of the calcium-sensitive dye results in high spatial resolution in opaque brain tissue2. Reconstruction of spikes from the fluorescence calcium recordings is achieved by a maximum-likelihood method. Simultaneous electrophysiological and optical recordings indicate that our method reliably detects spikes (&gt;97% spike detection efficiency), has a low rate of false positive spike detection (&lt; 0.003 spikes/sec), and a high temporal precision (about 3 msec) 3. This optical method of spike detection can be used to record neural activity in vitro and in anesthetized animals in vivo3,4.

Understanding the function of the vertebrate central nervous system requires recordings from many neurons because cortical function arises on the level of populations of neurons. Here we describe an optical method to record suprathreshold neural activity with single-cell and single-spike resolution, dithered random-access scanning. This method records somatic fluorescence calcium signals from up to 100 neurons with high temporal resolution. A maximum-likelihood algorithm deconvolves the underlying suprathreshold neural activity from the somatic fluorescence calcium signals. This method reliably detects spikes with high detection efficiency and a low rate of false positives and can be used to study neural populations in vitro and in vivo.

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also ...

Appetitive Associative Olfactory Learning in Drosophila Larvae

In the following we describe the methodological details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with genetic interference, provides a handle to analyze the neuronal and molecular fundamentals of specifically associative learning in a simple larval brain.
Organisms can use past experience to adjust present behavior. Such acquisition of behavioral potential can be defined as learning, and the physical bases of these potentials as memory traces1-4. Neuroscientists try to understand how these processes are organized in terms of molecular and neuronal changes in the brain by using a variety of methods in model organisms ranging from insects to vertebrates5,6. For such endeavors it is helpful to use model systems that are simple and experimentally accessible. The Drosophila larva has turned out to satisfy these demands based on the availability of robust behavioral assays, the existence of a variety of transgenic techniques and the elementary organization of the nervous system comprising only about 10,000 neurons (albeit with some concessions: cognitive limitations, few behavioral options, and richness of experience questionable)7-10.
Drosophila larvae can form associations between odors and appetitive gustatory reinforcement like sugar11-14. In a standard assay, established in the lab of B. Gerber, animals receive a two-odor reciprocal training: A first group of larvae is exposed to an odor A together with a gustatory reinforcer (sugar reward) and is subsequently exposed to an odor B without reinforcement 9. Meanwhile a second group of larvae receives reciprocal training while experiencing odor A without reinforcement and subsequently being exposed to odor B with reinforcement (sugar reward). In the following both groups are tested for their preference between the two odors. Relatively higher preferences for the rewarded odor reflect associative learning - presented as a performance index (PI). The conclusion regarding the associative nature of the performance index is compelling, because apart from the contingency between odors and tastants, other parameters, such as odor and reward exposure, passage of time and handling do not differ between the two groups9.

Appetitive Associative Olfactory Learning in Drosophila Larvae

Drosophila larvae are able to associate odor stimuli with gustatory reward. Here we describe a simple behavioral paradigm that allows the analysis of appetitive associative olfactory learning.

In the following we describe the methodological  details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with ...

Drosophila Adult Olfactory Shock Learning

Drosophila have been used in classical conditioning experiments for over 40 years, thus greatly facilitating our understanding of memory, including the elucidation of the molecular mechanisms involved in cognitive diseases1-7. Learning and memory can be assayed in larvae to study the effect of neurodevelopmental genes8-10 and in flies to measure the contribution of adult plasticity genes1-7. Furthermore, the short lifespan of Drosophila facilitates the analysis of genes mediating age-related memory impairment5,11-13. The availability of many inducible promoters that subdivide the Drosophila nervous system makes it possible to determine when and where a gene of interest is required for normal memory as well as relay of different aspects of the reinforcement signal3,4,14,16.
Studying memory in adult Drosophila allows for a detailed analysis of the behavior and circuitry involved and a measurement of long-term memory15-17. The length of the adult stage accommodates longer-term genetic, behavioral, dietary and pharmacological manipulations of memory, in addition to determining the effect of aging and neurodegenerative disease on memory3-6,11-13,15-21.
Classical conditioning is induced by the simultaneous presentation of a neutral odor cue (conditioned stimulus, CS+) and a reinforcement stimulus, e.g., an electric shock or sucrose, (unconditioned stimulus, US), that become associated with one another by the animal1,16. A second conditioned stimulus (CS-) is subsequently presented without the US. During the testing phase, Drosophila are simultaneously presented with CS+ and CS- odors. After the Drosophila are provided time to choose between the odors, the distribution of the animals is recorded. This procedure allows associative aversive or appetitive conditioning to be reliably measured without a bias introduced by the innate preference for either of the conditioned stimuli. Various control experiments are also performed to test whether all genotypes respond normally to odor and reinforcement alone.

Drosophila Adult Olfactory Shock Learning

The method to measure adult Drosophila associative memory is described. The assay is based on the ability of the fly to associate an odor presented with a negative reinforcer (electric shock) and then recall this information at a later time, allowing memory to be measured.

Drosophila have been used in classical conditioning experiments for over 40 years, thus greatly facilitating our understanding of memory, including the ...

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such behavior is spontaneous locomotor activity. Here we describe our protocol that utilizes Drosophila population monitors and a tracking system that allows continuous monitoring of the spontaneous locomotor activity of flies for several days at a time. This method is simple, reliable, and objective and can be used to examine the effects of aging, sex, changes in caloric content of food, addition of drugs, or genetic manipulations that mimic human diseases.

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster are useful in studying genetic or environmental manipulations that affect behaviors such as spontaneous locomotor activity.&#160; Here we describe a protocol that utilizes monitors with infrared beams and data analysis software to quantify spontaneous locomotor activity.&#160;

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Mass Histology to Quantify Neurodegeneration in Drosophila

Progressive neurodegenerative diseases like Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD) are an increasing threat to human health worldwide. Although mammalian models have provided important insights into the underlying mechanisms of pathogenicity, the complexity of mammalian systems together with their high costs are limiting their use. Therefore, the simple but well-established Drosophila model-system&#160;provides an alternative for investigating the molecular pathways that are affected in these diseases. Besides behavioral deficits, neurodegenerative diseases are characterized by histological phenotypes such as neuronal death and axonopathy. To quantify neuronal degeneration and to determine how it is affected by genetic and environmental factors, we use a histological approach that is based on measuring the vacuoles in adult fly brains. To minimize the effects of systematic error and to directly compare sections from control and experimental flies in one preparation, we use the &#39;collar&#39; method for paraffin sections. Neurodegeneration is then assessed by measuring the size and/or number of vacuoles that have developed in the fly brain. This can either be done by focusing on a specific region of interest or by analyzing the entire brain by obtaining serial sections that span the complete head. Therefore, this method allows one to measure not only severe degeneration but also relatively mild phenotypes that are only detectable in a few sections, as occurs during normal aging.

Mass Histology to Quantify Neurodegeneration in Drosophila

Drosophila is widely used as a model system to study neurodegeneration. This protocol describes a method by which degeneration, as determined by vacuole formation in the brain, can be quantified. It also minimizes effects due to the experimental procedure by processing and sectioning control and experimental flies as one sample.

Progressive neurodegenerative diseases like Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD) are an increasing threat to human health ...

Assessing Spatial Learning and Memory in Small Squamate Reptiles

Clinical research has leveraged a variety of paradigms to assess cognitive decline, commonly targeting spatial learning and memory abilities. However, interest in the cognitive processes of nonmodel species, typically within an ecological context, has also become an emerging field of study. In particular, interest in the cognitive processes in reptiles is growing although experimental studies on reptilian cognition are sparse. The few reptilian studies that have experimentally tested for spatial learning and memory have used rodent paradigms modified for use in reptiles. However, ecologically important aspects of the physiology and behavior of this taxonomic group must be taken into account when testing for spatially based cognition. Here, we describe modifications of the dry land Barnes maze and associated testing protocol that can improve performance when probing for spatial learning and memory ability in small squamate reptiles. The described paradigm and procedures were successfully used with male side-blotched lizards (Uta stansburiana), demonstrating that spatial learning and memory can be assessed in this taxonomic group with an ecologically relevant apparatus and protocol.

This paper describes a modification of the Barnes maze, a standard rodent paradigm used to assess spatial memory and learning, for use in small squamate reptiles.

Clinical research has leveraged a variety of paradigms to assess cognitive decline, commonly targeting spatial learning and memory abilities. However, ...

Drosophila Courtship Conditioning As a Measure of Learning and Memory

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model organisms such as the fruit fly, Drosophila melanogaster. Drosophila is useful for understanding the basic neurobiology underlying cognitive deficits resulting from mutations in genes associated with human cognitive disorders, such as intellectual disability (ID) and autism. This work describes a methodology for testing learning and memory using a classic paradigm in Drosophila known as courtship conditioning. Male flies court females using a distinct pattern of easily recognizable behaviors. Premated females are not receptive to mating and will reject the male&#39;s copulation attempts. In response to this rejection, male flies reduce their courtship behavior. This learned reduction in courtship behavior is measured over time, serving as an indicator of learning and memory. The basic numerical output of this assay is the courtship index (CI), which is defined as the percentage of time that a male spends courting during a 10 min interval. The learning index (LI) is the relative reduction of CI in flies that have been exposed to a premated female compared to na&#239;ve flies with no previous social encounters. For the statistical comparison of LIs between genotypes, a randomization test with bootstrapping is used. To illustrate how the assay can be used to address the role of a gene relating to learning and memory, the pan-neuronal knockdown of Dihydroxyacetone phosphate acyltransferase (Dhap-at) was characterized here. The human ortholog of Dhap-at, glyceronephosphate O-acyltransferase (GNPT), is involved in rhizomelic chondrodysplasia punctata type 2, an autosomal-recessive syndrome characterized by severe ID. Using the courtship conditioning assay, it was determined that Dhap-at is required for long-term memory, but not for short-term memory. This result serves as a basis for further investigation of the underlying molecular mechanisms.

Drosophila Courtship Conditioning As a Measure of Learning and Memory

This protocol describes a Drosophila learning and memory assay called courtship conditioning. This classic assay is based on a reduction of male courtship behavior after sexual rejection by a non-receptive premated female. This natural form of behavioral plasticity can be used to test learning, short-term memory, and long-term memory.

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model ...